A microscope according to the preamble and an autofocus method according to the preamble are known from WO 2007/144197 A1. This describes an autofocus device which images a modulation object that is intensity-modulated periodically along one direction into the sample and generates an autofocus signal from the image of the modulation object. Similar microscopes are found in DE 102006027836 A1 and DE 102007055530 A1.
This approach achieves very good autofocus properties, but occasionally has problems when the reflective properties of the sample vary locally, because then the image of the modulation object can be distorted by the reflection variations of the sample. In the worst case, the image of the modulation object can be altered by the object image so radically that an autofocus signal can no longer be generated.
DE 19537376 A1 uses two patterns from periodic grids, phase-shifted by 180°, in different focal planes. The patterns serve to determine direction for the autofocusing.
DE 102008005355 A1 describes the effect a reflectivity jump has on the AF contrast signal. To thereby avoid negative influences, a defocusing of the projected grating structures is proposed.
In U.S. Pat. No. 4,725,722, differently defocused grating images are subtracted from each other.